Antibiotic 21,190 RP

ABSTRACT

The new antibiotic 21,190 RP is prepared by cultivating the hitherto unknown microorganism Streptomyces hygroscopicus DS 23,230 (NRRL 3576) under aerobic conditions in an aqueous nutrient medium. The antibiotic is active against Gram positive and some Gram negative microorganisms. It can be used as a growth promoting agent for animals.

United States Patent Mancy et al.

July 1, 1975 ANTIBIOTIC 21,190 RP Inventors: Denise Mancy, Charenton;Jean Florent; Jean Preudllomme, both of Paris, all of France Assignee:Rhone-Poulenc, S.A., Paris, France Filed: Aug. 21, 1973 Appl. N0.:389,638

Related US. Application Data Division of Ser, No. 309,8l0, Nov. 27,I972, Pat. No. 3,822,350.

Foreign Application Priority Data Nov. 29, l97l France 7l.42683 US. Cl.424/118 Int. Cl. H61k 21/00 Field of Search 424/] 18 References CitedOTHER PUBLICATIONS Miller, The Pfizer Handbook of Microbial Metabolites,McGraw-Hill Book Co., N.Y., N.Y., l96l, pages 35, 40, I25, I26, 128 andS80.

Primary Examiner lerome D. Goldberg Attorney, Agent, or FirmStevens,Davis, Miller & Mosher [57] ABSTRACT 6 Claims, 2 Drawing Figures SHEETWAVE MJMBER (cm- 1 ANTIBIOTIC 21,190 RP This is a division ofapplication Ser. No. 309,810 filed, Nov. 27, 1972, now Pat. No.3,822,350.

This invention relates to a new antibiotic, hereinafter denoted by thenumber 21,190 RP, to its preparation by culture of a Streptomyces strainidentified more completely hereinafter and denoted by the nameStreptomyces hygroscopicus, DS 23,230 and to compositions containing theantibiotic.

A specimen of Streptomyces hygroscopicus DS 23,230 has been deposited atthe United States Department of Agriculture, Northern Regional ResearchLaboratory, at Peoria, 111., U.S.A., and has been given the number NRRL3576; a sample of the microorganism can be obtained from theaforementioned Research Laboratory.

Antibiotic 21,190 RP is of value because of the antimicrobial activitywhich it exerts principally against Gram-positive microorganisms, inparticular against staphylococci, as well as against certainGram-negative microorganisms, especially against bacteria of theNeisseria genus, in conjunction with its low toxicity.

Antibiotic 21,190 RP is characterised by the following physico-chemicalproperties:

Appearance: white crystalline powder.

Elementary Analysis: it contains carbon, hydrogen,

oxygen and chlorine in the following proportions:

C 50.5% H 6.6% O 38.4% and C1 4.5%

Solubility: it is soluble in water at pH 9 (10 g./l.) and indimethylformamide (50 g./l.), sparingly soluble in methanol and ethanoland practically insoluble in hexane and benzene.

Melting point: (determination on a Kofler block) Ultra-Violet Spectrum:when dissolved in aqueous 0.1N sodium hydroxide solution, 21,190 RPshows an absorption maximum at 292 nm (E 59) and an absorption minimumat 257 rim (E 33). FIG. 1 of the accompanying drawings shows the UV.spectrum of 21,190 RP dissolved in aqueous 0.1N sodium hydroxidesolution at a concentration of 50 pig/cc.

Infra-Red Spectrum: (determination from tablets of a mixture with KBr)this spectrum is shown in FIG. 2, in which the abscissae give thewavelength ex pressed in microns (lower scale) and the wave number in cm(upper scale) and the ordinate gives the optical density.

The principal infra-red absorption bands of 21,190

RP, expressed in wave numbers (cm"), are given in Table 1.

where \S \er strung: S strong; in medium. w Weak; \u \ery weak. shshoulder.

Optical rotation 1 3 1 3 (c 0.5, 0.1N NaOH) 1011 7 1* 3 (c 0.5, 0.1NNaOH) [011 20 1 3 (c 0.5, 0.1N NaOH) Colour reactions: antibiotic 21,190RP gives the following reactions:

Positive with the following reagents: cysteinecarbazole, sulphuric acid,Wheeler-Tollens, Bial, Fehling (after acid or alkaline hydrolysis) andMolisch.

Negative with Millon, Nessler, Ehrlich, Zimmerman-Bitto, Sakaguchi,Elson-Morgan and Dische reagents.

Chromatographic migrations:

The Rf-values for 21,190 RP, deposited in an amount of ug. on a plate ofKieselgel H (Merck) and revealed by bioautography with Staphylococcusaureus 209 P (ATCC 6538 P) as the sensitive microorganism, or byspraying with sulphuric acid and heating at 100C, are given in Table 11for some solvent systems.

TABLE I1 Solvents Rf Chloroforrn 0 Benzene 0 Benzene-methanol 90-10(v/v) 0.15 Benzene-methanol 80-20 [v/v) 0.60 Benzene-methanol 50-50(v/v) 0.87 Acetone 0.80 Methanol 0.85

Antibiotic Activity and Toxicity A. Bacteriostatic activity in vitro of21,190 RP The bacteriostatic activity of 21,190 RP against a certainnumber of microorganisms was determined by one of the dilution methodsusually employed for this purpose. For each microorganism the lowestconcentration of substance which, under defined conditions, prevents anyvisible development in a suitable nutrient broth was determined. Theresults of the various determinations are given in Table 111, whereinthe minimum bacteriostatic concentrations are expressed in micrograms ofsubstance per cc. of test medium.

TABLE 111 Minimum bacteriostatic concentrations (in pg/cc.)

Microorganisms tested Mvcobaclerium species ATCC 607 Escherichia coliATCC 9637 150 Shigella dysenrenae.

Shiga L (Institut Pasteur) 150 TABLE Ill-Continued Microorganisms testedMinimum bacteriostatic concentrations (in ag/cc.)

SEHnoneIIn paralyphr A (Lacasse strain. Institut Pasteur) 150 Salmonellasclmumuelleri (paratyphi B) Fougenc strain (Institut Pasteur) 150Proteus vulgarix 150 Klebsiella puetmtoniae ATCC 10031 150 Pseudomonasueruginuxa (Bass strain, lnstitut Pasteur) 150 Bruceila broncliiseprica(CN 387 Wellcome Institute) 35 Pusteurella mulrocida (A 125. InstitutPasteur) 0.2 Mycoplasma galll'sepricum (A 514, Institut Pasteur) 321,190 RP also possesses a marked bactericidal ac tivity, in vitro,against staphylococcus; the ratio be tween the bacteriostatic andbactericidal concentrations is of the same order as that of penicillinG.

B. Toxicity The toxicity of 21,190 RP is low as is shown by the resultsobtained by subcutaneous administration to mice:

LD 2,500 mg./kg. animal body weight administered subcutaneously LD 1,000mg./kg. animal body weight administered subcutaneously.

C. Antimicrobial activity in vivo Antibiotic 21,190 RP is active, inmice, against experimentally-induced infections caused by staphylo'cocci, streptococci and meningococci, when it is administeredsubcutaneously, but is inactive when admin' istered orally. When theproduct is administered subcutaneously for two consecutive days to mice,the 50% curative doses (CD are between 2 and 18 mg./kg. animal bodyweight, administered subcutaneously, de pending on the bacterium used.

The organism which produces antibiotic 21,190 RP is a strain ofmicroorganism which has been isolated from a sample of earth taken inGreat Britain, in Middlesex, and to which the number DS 23,230 (NRRL3576) has been given.

The isolation of this microorganism was carried out by following thegeneral method which consists of suspending a small amount of earth insterile distilled wa ter, diluting the suspension to differentconcentrations, and spreading a small volume of each dilution on thesurface of Petri dishes containing a nutrient agar medium. Afterincubation for several days at 26C., which makes it possible for themicroorganisms to develop, the colonies which it is desired to isolatein order to continue the investigation of them are removed andtransplanted to sloping nutrient agars for the purpose of producing moreabundant cultures of them.

The strain of microorganism DS 23,230 belongs to the Streptomyces genusand, more precisely, is related to the species Strepmmyceshygroscopicus, the essential characteristics of which have been definedby H. D. Tresner and E. J. Backus (Applied Microbiology, 4, 243-250,1956) and by S. A. Waksman (The Actino mycetes, ll, The Williams andWilkins Company, Baltimore, 1961, pages 230-231 This is why it has beencalled Streptomyces hygroscopicus, DS 23,230.

S. hygroscopicus, DS 23,230 possesses, in effect, the following threeproperties which correspond to the three characteristics by which H. D.Tresner and E. .l. Backus as well as S. A. Waksman define the species 5.hygmscopicus: (21) its sporiferous filaments generally terminate inclosed spirals with a coil of a few turns; these sporiferous filamentsare usually inserted along a main filament, forming clusters which maybe more or less elongated; (b) when its sporulated aerial mycelium hasreached a good stage of development, it shows a dark grey colourationcorresponding to that shown by the species S. hygrosc'opicus, and (c) oncertain culture media which make good sporulation possible, after aging, shiny black regions with a wet appearance appear in the sporulatedsurfaces, characteristic of the species 5. hygroscopicus; in the case ofS. hygroscopicus, DS 23,230, the conversion of the dark grey sporulatedaerial mycelium into a black coating takes place only in quite aninconspicuous way, which is in general limited to small points or smallregions distributed over the sporulated surface rather than taking placeover this entire surface area. The appearance of these black regions ishowever evident and can be observed in particular on Hickey and Tresneragar, Pridham yeast extract agar, Pridham oatmeal and tomato agar,glucoseasparagine agar, starch-nitrate agar and Pridham starch inorganicsalts agar.

H. D. Tresner and E. J. Backus describe the production of awine-coloured soluble pigment, on certain media, varying according tothe particular case, by certain strains of the species S. hygruscopicus.The strain DS 23,230 possesses this property to a very marked extent; itproduces a reddish pink soluble pigment which may be more or lessviolet-coloured on quite a large number of media, and in such a way thatin many cases it is capable of colouring the aerial mycelium which thenassumes a pink shade before showing the characteristic grey colourationwhich it has when sporulation takes place.

On the few culture media where its morphological appearance isdescribed, the strain S. hygroscopicus mentioned as reference by S. A.Waksman in The Actinomycetes shows a certain number of differencescompared with the strain DS 23,230. The most noticeable are that it doesnot give any soluble pigment on gelatine, that it produces a lightyellow soluble pigment on glucose-asparagine agar, and that it gives awell developed culture on nitrate agar containing sucrose, whilst thestrain DS 23,230 gives a dark orange-brown soluble pigment on gelatine,as well as on asparagineglucose agar and, due to the fact that it doesnot utilise sucrose, does not develop on Czapek synthetic nitrate agarcontaining sucrose. Furthermore, in no case is it stated that itsnon-sporulated aerial mycelium is coloured pink, as has just been statedin the case of the strain DS 23,230. However, these several differencesare not sufficiently important, according to the ideas of H. D. Tresnerand E. J. Backus, for it to be considered that l the strain DS 23,230can constitute a species which is different from the species S.hygroscopicus, since it presents the main characteristics of S.hygroscopicus which serve to define it.

S. hygroscopicus, DS 23,230 forms sporiferous filaments which generallyterminate in closed spirals containing 1 to 5 turns, although veryoccasionally spirals forming a larger number of turns, or somesporiferous filaments which are simply curved over at their end partwithout forming a complete turn, or sometimes spirals which may be moreor less loose and unwound,

are observedv The sporiferous apparatus possesses a cluster structure.the spiral sporiferous filaments. which themselves can show somebranches. being inserted along a main filament which can be quite long.The spores are oval and measure 0.6 to 0.8/1.0 to l.2;.t. Microscopicexaminations have shown an identical arrangement of the sporiferousapparatus on Hickey and Tresner agar and on Pridham starch-inorganicsalts agar.

The culture characteristics and the biochemical properties of S.liygrosc'upicus, DS 23.230 are given in Table IV which follows. Unlessotherwise stated. they are those of cultures which have reached a goodstage of development, having been aged for about 3 to 4 weeks at 26C.These properties have been observed on nutrient agars and broths usuallyemployed to determine the morphological properties of strains of Strep'tomyces, the cultures on agar media being carried out on agar slopes. Acertain number of culture media used were prepared in accordance withformulations indicated in The Actinomycetes" (S. A. Waksman, pp.l93-l97, Chronica Botanica Company, Waltham. Mass, U.S.A., 1950); inthis case, they are indicated by the letter W followed by the numberwhich was given Ref. G melanin formation medium The Actino mycetes. vol.2. p 333 No. 42 S. A. Waksman The Williams and Wilkins Company.Baltimore. 1961 Ref. H W. E. Grundy et coll Antibiotics and Chem., 2.401. I952 Refv I Inorganic Salts Starch Agar T. G. Prid ham ct collAntibiotics Annual. 156-57. p. 951

Ref. J Corresponds to the formulation W l wherein g. of sucrose arereplaced by 15 g. of glucose Ref. K Corresponds to the formulation W- lwherein 30 g. of sucrose are replaced by l5 g. of glyccrine Ref. L Plaingelatin 1 prepared according to the instructions in "Manual of Methodsfor Pure Culture Study of Bacteria of the Society of AmericanBacteriologists Geneva. N.Y.. H -l8 Ref. M Manual of Methods for PureCulture Study of Bacteria of the Society of American BacteriologistsGeneva. N.Y., 18

Ref. N Synthetic medium of Dimmick" (not containing agar) Manual ofMethods for Pure Cul ture Study of Bacteriaof the Society of AmericanBacteriologists. Geneva. N.Y.. ll -,.,l9

. is them 1n The Actinomycetes The references or con- 0 correspondS tothe formulamm W4 8 stitutions of the other culture media are as followszwherein 30 g of Sucrosg are repklcgd by 15 0f Ref. A. Hickey andTresners Agar T. G. Pridglucose i at Alymblongs l 125951 R 950 Ref PCorresponds to the formulation W-l8. B Sgrmulauon to whlch of agar has 0wherein the sucrose is omitted and replaced by been a cd small strips offilter paper partially immersed in the Ref. C K. L. Jones Journal ofBacteriology, 57. liquid 142, 1949 R f D Yeast Extract Agar" 1 pridhamct CO" Ref. 0 Commercially available skimmed milk pow- AntibioticsAnnual 95 57 950 der. reconstituted in accordance with the manufac- Ref.E Tomato Paste Oatmeal Agar" T. G. Pridmstructlons ham Ci CollAntibiotics Annual 1 P 950 Ref. R Medium indicated for the research ofthe R f F Pepto mkal extract (13% Y production of H 5 by: H. D. Tresnerand F. Danga sin -57t ag Journal of Bacteriology, 76. 239-244. l958.

TABLE IV Culture media Degree of Vegetative Aerial apparatus (com-Soluble Observations and biode elopniy'celium prising the combinationpigment chemical properties merit (V.m.] or of the aerial myceliumunderside of and the sporulation) the culture Hickey and Good UndersidcPink to pink-grey and Light Sporiferous apparatus Tresner agar lightorangedark grey. with points orange in clustersv Sporife- (Ref. A) brownhaving the black appearbrown rous filaments ending anee characteristicof in closed spirals of Hy-groscopicus" l to 5 turns. O al sporesmeasuring 0.6 to till/Ill to L2 p. Emerson agar Very undersidePink-vvhite Oraiige- (Ref. Bl good orangebrown brown Bennett agar VeryUnderside Light pink-grey Orange (Ref. Cl good dark orangebrown brownPridham yeast Very Underside Greyishpink to light Very extract agar gooddark orangcgrey and dark grey. dark (Ref. D) brown with points havingthe orange black appearance brown characteristic of Hygroscopicus"Pridham Very Underside Grcyish-pink to grey. Very dark oatmeal and goodblackwith many points having orangetomaio agar brown the blackappearance brown. rang- (Ref. E) characteristic of ing towards"Hygroscoplcu? blackish Glucose- Good Underside Light grcyish-pink toSlightly pepionc agar violetlight violet-grey violet- (W-7| browncoloured brown Nutrient agar Moderate Underside Greyish-whitc. None (W5|vcllou Poorly developed Nutrient agar Quite Underside Whitish LightSolubilisation of Containing good light brownycllowtyrosine. positive t\rosinc yellow bro n TABLE IV Continued Culture media Degree ofVegetative Aerial apparatus lcom- Soluhle Ohsenations andhimdevelopmycclium prising the comhination pigment chemical propertiesment (V.m.| or of the aerial myceliuni underside of and the sporulationlthe culture (Ref. F)

Tyrosine-yeast Quite Underside Greyish-white Very light Production ofmelanin:

extract agar good yellowishbrownish negative (readings taken {"MelaninForwhite according to the author's mation medium" reeoinmendationsl ofWaksmanl tRefr G) Krainsky cal- Extremely V.l|l colour- None NoneSoluhilisation oi the cium malate poor less to malate: none or agarwhitish. in extremely low (Ref. H) trace amounts Ovalhumin agar VeryV.m. orange- None Orange- (W- l 2) moderate brown brown Glucose- QuiteUnderside Light pink'grey to light 'ery dark asparagine good dark browngrey and dark grey. with orange agar a few small points havbrown (W-Z)ing the black appearance characteristic of Hygroscopicus" Glycerine-Good Underside Greyish-orange to light Blackishasparagine very darkgrey. Smali droplets of brown agar brown yellow-brown exudation Starch-Very Underside Pink-white to violetgrey Light pinlt- Hydrolysis of thestarch.

nitrate agar moderate very dark with a few small points violet positivetW-lOJ violet having the black appearance characteristic ofHygroscopicus" Pridham Very good Underside Greyish-white to lightGreyish- Sporiferous apparatus in starchdark ycllovvgrey and dark grey.yellowclusters. Sporiferous inorganic brown with points having the brownfilaments ending in closed salts agar black appearance spirals of l to 5turns.

(Ref. ll characteristic of Oval spores measuring (to "Hygroscopicus" to(LS/ll) to l2 a Hydrolysis of the starch. positive Czapek Practic- Nonesynthetic ally agar containnone ing sucrose Czapek Good UndersideGreyish-pink violet Very dark synthetic very dark Exudation of a fewsmall reddishagar containreddishpink droplets \tolet ing glucose violet(Ref. J)

Czapek Very Underside Violet-pink. Esudation Very dark synthetic goodvery dark of small pink droplets reddish agar containred-brown violetingglycerine fl n (Ref. K)

Potato Very V.m. very Greyishwvhite tinged Orangeculture (W-Z?) goodwell devel light pink in places. brown to oped and Numerous droplets ofdark violetvery wrinkled; light brownish-yellow to reddish completely;orange-brown exudation brown covered by the aerial mycelium l2 r pureGood Well developed Whitish. Moderately Orange- Liquefaction of thegelatine surface developed brown gclatinc: quite rapid (Ref. Ll culture.

Underside light orangebrown Nutrient Moderate Thin. whitish v'crymoderately None Production ol'nitrites nitrate brownishdeveloped fromnitratesaiegati e broth (Rel'.M) yellow ring Starch nitrate AverageWhitish ring Whitish. Very moderately Very vveak Production of nitritesbroth [W-IQ) and velum developed brownish from nitrateszpositive DimmickModerate Small Whitishl In trace Very weak Production of nitritesglucose yellowishamounts brownish from nitratexpositive nitrate brown atthe start of the broth tRel'N) colonies at culture, but quite thesurface quickly becomes negative of the culture Czapek Moderate Smallwhitish Whitisl'i Very moderately None synthetic colonies rlevelorwlbroth contain clustered toing glucose gether at the lRefO) surface ofthe culture Czapek None Utilisation of the synthetic cellulose. negativebroth containing cellu lose (Rel. P)

TABLE IV Continued ('ulturc media Degree of Vegetative Aerial apparatus(eom' Soluble Observations and biodevelop mycelium prising thecombination pigment chemical properties ment (V.m.l or ofthe aerialmycelium underside of and the sporulation) the culture Sktmmed PoorPeptonisation without milk (RefQl coagulation,

ph unchanged in 1 month Tresncr and Quite \Ymi light None Very lightProduction of H. ,S: Danga agar good yellowishyellowish ncgathc(readings for investigbrown brown taken according to the ation of theauthor's recommendations) production of H 3 (Ref, R)

The capacity of Slreprmnycex hygrosmpicux DS 23.230 for using varioussources of carbon and nitrogen to achieve its development has beendetermined according to the principle of the Pridham and Gottlieb used.In particular the following sequence of operations can be adopted:

Streptomyces hygroscopictu'. DS 23.230 stock 20 method (J. of Bact. 56.lO7-l l4, 1943), the degree of culwrelon agar development was observed.after a suitable incubation period at 26C. on the base medium indicatedby the Culture in an dgimted flask authors, by replacing either theglucose by the various sources of carbon respectively tested, or (NH SOby N i h Culture in a fern-enter the various sources ofnitrogenrespectively tested. The 1 results are given in Table V. productlonculture in a fermentcr TABLE V Sources of Utilisation Sources ofUtilisation carbon nitrogen tested tcstcd D-Ribose positive NaNOpositive D-Xy lose positive NaNO positive L-Arubinosc negative (NHQ SOpositive L-Rhamnosc negative INHU HPO positive D-Glucose positiveAdenine positive D-Galactose positive Adenosinc positive DFructosepositive but vveak Uracil negative D-Mannose positive Urea positiveL-Sorhose negative L-Asparagine positive Lactose positive Glycinepositive Maltosc positi\e Sarcosinc negative Sucrose negative DL-Alanincpositive Trchulosc positi\c DL-Valine positive Cellobiosc negativeDL-Aspartic positive acid Raftinose positive L-Glutamic positive acidDcvtrin posith e L-Arginine positive lnulin negative L Lysine positiveStarch positive DL-Scrinc pusitiu: Glycogen positive DL Threonincpositive Glycerol posithe DL-Methioninc negative Erythritol negativeTaurinc negative Adonitol negative LTyrosine positive Dulcitol negative[)L-Proline positive UMannitol positive L-Hydroxypositive prolineD-Sorbitol negative L-Histidine positive lnositol positiu: Salicincncgathc According to a feature of the invention the antibiotic 2 l ,lQORP is produced by aerobically cultivating Strep- I(JHI hvermt'opicus DS23.230 (NRRL 357(1), or a mutant thereof capable of producing theantiobiotic, using an aqueous nutrient medium containing assimilablcsources of carbon, nitrogen and inorganic substances and isolating fromthe medium 2l.l9() RP formed during the culture The culture ofStrepmnrvces liygruscripit'us DS 23,230 can be carried out by any of theknown aerobic surface culture or submerged culture methods, but thelatter are preferred for reasons of convenience. For this pur pose. thevarious types of apparatus which are currently employed in thefermentation industry may be The fermentation medium must contain anassimilable source of carbon and an assimilable source of nitroerol ormannitol, or certain organic acids c.g. lactic acid or citric acid.Certain animal or vegetable oils such as lard oil or soya bean oil maybe advantageously used gen, inorganic substances. particularlychlorides. and optionally growth-promoting factors; all theseingredients may be supplied as well-defined products or com plexmixtures such as those found in biological prod acts of various originsAs sources of assimilable carbon, there may be used carbohydrates suchas glucose, maltose dextrins, starch, or other carbon-. hydrogen andoxygencontaining substances such as sugar alcohols c.g. glycinstead of.or in admixture with. the aforementioned carbon. hydrogenandoxygen-containing substances.

The suitable sources of assimilable nitrogen are extremely varied. Theycan be very simple chemical compoudns such as inorganic or organicammonium salts. urea or certain amino acids. They can also be complexsubstances containing principally nitrogen in a protein form. such ascasein. lactalbumin. gluten and their by drolysates. soya bean flour.peanut meal. fish meal. meat extract. yeast extract. distillers solublesor cornsteep liquor.

Amongst the inorganic substances. some may have a buffering orneutralising effect such as the alkali metal or alkaline earth metalphosphates or the carbonates of calcium or magnesium. Others contributeto the ionic equilibrium necessary for the development of Srrepromycesliygmxcopicu.r. DS 23.230 and for the production of 21 .l90 RP. such asthe chlorides and sulphates of the alkali metals and alkaline earthmetals. Some of them act more especially as activators of the metabolismof Streptomyces IITgIOSCOPiCLLl'. DS 23.230. cg. the salts of zinc.cobalt. iron. copper and manganese.

The pH of the fermentation medium at the start of the culture should bebetween 6.0 and 7.8 and preferably between 6.5 and 7.5. The optimumfermentation temperature is 2530C.. but satisfactory production isachieved at temperatures between 23 and 33C. The rate of aeration of thefermentation broth can vary within quite wide limits, but it has beenfound that aera tion rates of 0.3 to 3 litres of air per litre of brothper minute are particularly suitable. The maximum yield of antibiotic 2l .l90 RF is obtained after 2 to 8 days culture. but this period dependspredominantly on the medium used.

It can be seen from the foregoing that the general conditions for theculture of Streptrmzyces' Irvgroscopr'cus. DS 23.230 for the productionof 21.190 RP can vary to quite a large extent and can be adapted foreach particular requirement.

2l.l90 RP can be isolated from the fermentation broth in the followingmanner.

The antibiotic is extracted from the filtrate of the fermentation brothby water-immiscible solvents. such as aliphatic alcohols having at least4 carbon atoms (e.g. butanol). chlorinated hydrocarbons (e.g. methylenechloride). esters (e.g. ethyl acetate) and ketones (e.g. methyl isobutylketone). This operation can be carried out at a pH between 3 and 9. andpreferably at about pH 7.

After decantation. the crude antibiotic can be iso' lated from itssolution in the above-mentioned organic solvents by concentration ofthese solutions under reduced pressure. followed by precipitation by anonsolvent or a poor solvent, for the antibiotic. such hexane. or byallowing to stand in a cold chamber.

The crude 2l.19() RP can be purified by conventional methods such asrecrystallisation. chromatogra phy on various adsorbing agents orcounter-current distribution.

lt is advantageous to purify 21.190 RP by recrystalli sation fromethanol after treatment with activated charcoal. followed byrecrystallisation from a mixture of dimethylformamide and water.

The following Example illustrates the invention.

In the following. the activity is always determined by means of themicrobiological diffusion method. using Slaphylococcux aureus' 209 P(ATCC 6538 P) as the sensitive microorganism. by comparison with asample of pure 21.190 RP taken as the standard and containing L000ig/mg. This activity is expressed in [Lg/CC. for solutions and in pg/mg.for solid products.

EXAMPLE 1 A l7(]litre fermenter is charged with:

peptone tCl 3.5%) [.200 g. rncat extract (CI Wt) 600 g, (crelosc L200 g.agar 240 g. tap water. sufficient to make up to l 10 litres The pH isadjusted to 7.20 with ION sodium hydroxide solution 120 cc. The mediumis sterilised by bubbling steam at [22C. through it for 40 minutes.After cooling. the volume of the broth is l20 litres and the pH is 6.60.The broth is inoculated with a culture (200 cc.) of Srreprrmryceshygroscopicus, DS 23,230 in a shaken Erlenmeyer flask. The culture isdeveloped at 26C. for 29 hours with agitation and aeration with sterileair; it is then suitable for inoculating the produc' tion culture.

The production culture is carried out in a 800litre cobalt chloride. 6 H0 8 g. tap water. sufficient to make up to 325 litres The pH is adjustedto 7.80 with lON sodium hydroxide solution 1.200 cc. and the medium issterilised by bubbling steam at l22C. for 40 minutes. After cooling. thevolume of the broth is 355 litres. it is made up to 400 litres by addingan aqueous sterile solution (40 litres) containing hydrated glucose (8kg.) and an aqueous sterile solution (5 litres) containing ammoniumsulphate (800 g.) The pH is then 6.80.

The broth is then inoculated with the inoculum culture (40 litres) fromthe l-litre fermcnter described above. The culture is carried out at26C. for 64 hours with agitation using a motor rotating at 205revolutions per minute and aeration with sterile air (20 m lhour).

After 24 and 48 hours of culture. an aqueous sterile solution (5 litres)is added. containing hydrated glucose (2 kg).

At the end of the operation, the pH of the culture is 7.50 and thevolume of the broth is 410 litres. The amount of 21.190 RP present is240 [Lg/CC.

The broth is introduced into a tank equipped with a stirrer. and afiltration aid (Clarcel DIC) (30 kg.) is then added. The suspension isfiltered on a filter press and the filter cake is washed with waterlitres). The filtrate. the volume of which is 430 litres. is extractedin two stages with butanol (240 litres). using a group of centrifuges incountercurrent. The flow rate of the filtrate is adjusted to litres/hourand the flow rate of the solvent to 100 litres/hour. The extract. thevolume of which is 250 litres. contains the major portion of theactivity. It is washed with water (25 litres). The washed extract has avolume of 225 litres. lt is concentrated at 35C. under reduced pressure(20 mmHg) to a volume of 2 litres.

The concentrate is left to stand in a cold chamber at 5C. After 12 hoursat this temperature the precipitate which has formed is filtered off.washed with butanol (500 cc.) and dried. Antibiotic 21.190 RP (230 g.the strength of which is 260 ugjmg. is thus obtained.

Crude 21,190 RP 137 g.). prepared under the above conditions, but ofstrength 216 peg/mg. is suspended in ethanol (35 litres). The suspensionis heated under reflux for 30 minutes and then filtered whilst hot. Theinsoluble material is discarded. The filtrate. to which decolourisingcharcoal (Darco G 60) g.) has been added, is stirred whilst heatingunder reflux for 15 minutes and then filtered using a filtration aid (10g).

The solution obtained is concentrated under reduced pressure to a volumeof 1 litre. The concentrate is placed in a cold chamber at 5C. for 12hours. After this period. the crystals which have formed are filteredoff. washed with ethanol cc.) and dried. Crystalline 21,190 RP (26.2 g.)of which the strength is 920 ag/mg. is thus obtained.

The antibiotic (85.5 g.), prepared under the above conditions, isdissolved in dimethylformamide (4.25 litres). The solution is clarifiedby filtration and then introduced into a flask equipped with a stirrer.Water (3 litres) is run slowly into the clarified solution, whilstcontinuing the stirring. After the end of the addition, the suspensionof crystals is cooled to 10C. The crystals are then filtered off, washedwith a dimethylformamide/water (1:1 v/v) mixture (1 litre) and then resuspended in water 1.5 litres). They are finally filtered off and driedfor 24 hours at 40C. under reduced pressure (1 mm.Hg). Crystalline21.190 RP (61.5 g.), of strength 1000 p.g./mg.. melting at 226-228C., isthus obtained.

Since antibiotic 2 l ,190 RP is particularly active against streptococciand staphylococci, it can advantageously be used in the local treatmentof mastitis, metritis and cutaneous infections of domestic animals. At adose of 1 mg./kg. animal body weight (intramuscular) the antibioticshowed good activity in chickens infected with Borrelia unsvrina(responsible for avian spirochaetes) The present invention includeswithin its scope therapeutic compositions for veterinary use comprising21.190 RP in association with a physiologically acceptable carrierand/or a compound which may itself be physiologically active. forexample an antimicrobial agent such as an antibiotic with a differentantibacterial spectrum, or an antifungal agent.

For therapeutic application. antibiotic 21.190 RP can be made up in anyof the usual various forms suitable for the method of administrationenvisaged. such as liquid formulations (suspensions. or dispersions,preferably prepared at the time of use from a solid composition). pasteformulations (creams and ointments) and solid formulations (vaginaltablets. ovules and powders). The proportion of 21,190 RP may be variedaccording to the method oftreatment and of the type of formulation. Thusa formulation prepared immediately before use for intramuscularinjection can comprise a unit dose of 21.190 RP and a dose of liquidsuitable for veterinary use. for example an isotonic saline solutionbuffered to pH 7.2 with phosphates, to give a suspension containing 1%by weight of antibiotic. A powder for dusting can contain 5 to 507: by

weight of 21.190 RP. a powder which can be dispersed in a liquidsuitable for intramammary injection can contain up to 99% by weight ofantibiotic in association with a physiologically acceptable dyestuffsuch Blue F.C.F. (Alphazurine FG Food Blue No. 2 C]. No. 42.090) andwith a physiologically acceptable dispersing agent, and an ointment cancontain 0.5 to 10% by weight of antibiotic. the excipients being chosenfrom amongst those which are usually employed in these types offormulations.

When added to animal feedstuffs antibiotic 21.190 RP makes it possibleto achieve an increase in the weight of the animal which is more rapidthan with feedstuffs devoid of it.

Another aspect of the present invention therefore comprises animalfeedstuffs, or concentrated mixtures for animal feeding, containing theantibiotic 21.190 RP.

The dose necessary to produce a suitable growthpromoting effect cannaturally vary within quite wide limits depending on the species ofanimals and on the nutritional value of the feedstuffs themselves. 1ngeneral terms, it is sufficient for the feed rations made available tothe animals to contain 1 to 50 g. of antibiotic 21.190 RP per metrictone of l'eedstuff (and up to g. per metric ton for weaner feedstuffs).

The antibiotic 21.190 RP can be contained as a uniform dispersion incomplete composite feedstuffs. at the above doses.

It can be distributed in supplementary feedstuffs to an extent of 0.1 to0.000l7( by weight thereof. most frequently with other additives such asvitamins and inorganic salts. These supplementary feedstuffs can ci therbe mixed with the animals rations or consumed directly. and usuallyrepresent about 5 to 20% of the ration.

The premixes, which are used for preparing complete rations orsupplementary feedstuffs. usually contain 0.05 to 20% by weight ofantibiotic 21,190 RP diluted with a physiologically innocuous carrier.for example. an amount of food. They constitute a convenientintermediate which makes it easier to distribute the antibiotic 21,190RP evenly in feedstuffs. The premixes themselves are generally producedfrom concentrates which contain 99.9 to 20% by weight of antibiotic21,190 RP. to which a physiologically innocuous carrier or edibledenaturants such as feed dyestuffs. flavouring agents. dispersing agentsor agents which prevent agglomeration. and fillers. have been added. Aconcentrate can contain. for example, by weight, 99% of antibiotic21.190 RP with 0.1% of dyestuff and 0.9% of anti-agglomerating agent.

The concentrates and prcmixes are generally pow ders. The supplementaryfeedstuffs and the complete composite feedstuffs can be either powdersor in the form of granules, prepared according to the usual techniques.In these different compositions. antibiotic 21,190 RP can be in the formof fine free particles or covered with a coating.

The antibiotic 21,190 RP is suitable for administration to all animalsand especially to fowls.

The following Examples illustrate this aspect of the invention.

EXAMPLE 2 Chicks (SELAF 915) are distributed in batteries at the rate of14 per coop. each coop containing chicks of EXAMPLE 3 The procedure ofExample 2 is followed. but using a basic foodstuff having the followingcomposition:

The chicks are weighed and the amount of feedstuff wheat flour Wconsumed is determined after 2 and 4 weeks. The gain mi 'f n l in weightand the consumption index is calculated after 503x11 bean flour s. 2weeks and after 4 weeks. 1

. I I I dried m||k powder 11" The chicks receive a bdSlC feedstuffhaving the foldimmers MUN 2Q l i iti 1 mineral and \itamin comp1e\ 40,

z 5 3, 2352;513: p 1: This foodstuif contains 21% of digestible protems.maize flour 13.41% The vitamin contents. calculated per 100 kg. are aswheat flour 31.32 1 follows. fish meal 8.92% i soya bean Hour 8.29% 15vitamin A 1.400.000 1L dehydrated ha flour 4.56% \itamin D; 401,500 1Uyeast solids (whole cells or autolysate: 2.33?! vitamin B 00 mg driedmilk powder 2.68% im i B2 7 mg. sodium chloride 0.09% vitamin B, 1.600mg. calcium carbonate 0.89% ilun in B' 300 mg mineral elements (Mn. 1.and Zn) 0.0692 i i mg This fcedstuff contains in addition. 20 vitamin C1.000 mg. v tam n A 4.000 lU/kg. vitamin E 2.700 mg. vitamin D 1,000lU/kg. vitamin K, 125 choline chloride 11.5 mgJkg. \immin pp (L200 mgriboflavin 2.24 mgJkg. r n acid 35 mg biotin 10 mg. t h I Q I The chicksare divided mto 2 groups (4 coops per c 17mm) group. each groupcontaining 28 young hens and 28 The chickens are divided into 2 groups.Group 1 re young cocks). Group 1 receives the control feedstuff. ceivesthe control feedstuff. Group 11 receives the con Group 11 receives thecontrol feedstuff to whch the antrol feedstuff to which the antibiotic21,190 RP has tibiotic 21.190 RP has been added at a concentration beenadded at a concentration of 10 g. per metric ton of 10 g. per metric tonof feedstuff. m of feedstuff.

The results obtained are as follows: The results obtained are asfollows:

1 Average weight (in grams) on day 1 at 2 weeks at 4 weeks young youngyoung cocks cocks cocks young young and young young and young young andcocks hens young cocks hens young cocks hens young hens hens hens Group1 38 37 37 189 181 185 510 452 479 Group 11 37 37 37 202 193 197 559 494527 11 Gains in weight and consumption indices Group 1 (control) Group11 young young cocks cocks young young and young young and cocks hensyoung cocks hens young hens hens 1 day to Gain in weight (in g.) 151 144148 165 156 160 2 weeks Consumption index 1.56 1.67 1.62 1.60 1.62 1.61

2 weeks to Gain in weight (in g.) 321 271 294 357 301 330 4 weeksConsumption index 1.98 2.2 2.09 1.85 2.03 1.93

1 day to Gain in weight (in g.) 472 415 442 522 457 4 10 4 weeksConsumption index 1.84 2.02 l 93 1.77 1 89 1.83

Gain in weight (in ft 100 100 110.4 110 relative to the control]Consumption index (in i2 100 100 100 96.192 93.591 94.8% relative to thecontrol) 1 Average Weight (in grams] ay l at 2 weeks at 4 weeks youngyoung young cocks cocks cocks young young and young young and youngyoung and cocks hens young cocks hens young cocks hens young hens henshens Group 1 36 35 3a 206 194 200 639 582 610 Group 11 3o .15 35 2122113 207 6612 (i211 6141 17 18 ll Gain in weight and Consumption IndexGroup 1 (control) Group 11 young young cocks cocks young young and youngyoung and cocks hens young cocks hens young hens hens 1 day to Gain inweight (in g.) 170 159 164 176 168 172 2 weeks Consumption index 1.351.35 1.35 1.35 1.32 1.33

2 weeks to Gain in weight (in g. 433 388 410 450 417 434 4 weeksConsumption index 1.92 1.93 1.93 1,83 1.90 1.86

1 day to Gain in weight (in g.) 603 547 574 626 585 606 4 weeksConsumption index 1.76 1.76 1.76 1.69 1.73 1.71

Gain in weight (in Z relative to the control) 100 100 100 100.8 106.9105.6 Consumption index (in Z relative to the Control) 100 100 100 9698.3 97.1

We claim: 3,440 strong, 2,970 medium, 2,930 strong, 2,900

1. A feedstuff composition comprising an animal feedstuff and 0.1 to0.00017r by weight ofsaid feedstuff of the antibiotic herein designated21, 190 RP, which possesses the following characteristics: it is a whitecrystalline powder, melting at 226228C., soluble in water at pH 9 and indimethylformamide, sparingly soluble in methanol and ethanol, andpractically insoluble in hexane and benzene; it contains carbon,hydrogen, oxygen and chlorine and has the elementary composition: C50.5% H 6.6% O 38.4% C1= 4.5%; its ultra-violet spectrum in aqueous 0.1Nsodium hydroxide solution shows an absorption maximum at 292 nm (E, H33) and an absorption minimum at 257 nm (E, 33); its infra-red spectrum(determined with a mixture with potassium bromide) shows principalabsorption bands as follows: 3,440 strong, 2,970 medium, 2,9300 strong,2,900 shoulder, 2,840 shoulder, 1,735 strong. 1,715 medium, 1,630medium, 1,570 medium, 1,335 shoulder, 1,305 shoulder, 1,248 strong,1,220 weak, 1,192 medium, 1,165 medium, 1,122 strong, 1,090 strong,1,060 strong, 1,035 very strong, 975 medium, 942 medium, 920 shoulder,900 weak, 865 me dium, 848 weak, 828 weak, 815 weak, 775 medium, 730medium, 690 weak, 678 very weak, 655 weak, 630 shoulder, 612 weak, 570weak, 548 weak, 525 very weak, 510 very weak; and its optical rotationis 1121],," 3 3 (c 0,5, 0.1N NaOH), [01] +7 1 3 (c 0.5, 0,1N NaOH),[a],,,,,,*"= 3 (0 0.5, 0.1N NaOl-l).

2. A feedstuff according to claim 1, comprising 1 to 80 g. of 21.190 RPper metric ton of said feedstuff.

3. A premix for an animal feedstuff which comprises, in association withan animal fecdstuff or a physiologically innocuous carrier, theantibiotic herein designated 21 ,190 RP which possesses the followingcharacteristics: it is a white crystalline powder, melting at 226228C.,soluble in water at pH 9 and in dimethylformamide, sparingly soluble inmethanol and ethanol, and practically insoluble in hexane and benzene;it contains carbon. hydrogen, oxygen and chlorine and has the elementarycomposition: C 50.5% H 6.6% O 38.4% C1 4.5%, its ultra-violet spectrumin aqueous 0.1N sodium hydroxide solution shows an absorption maximum at292 nm (E, 59) and an absorption minimum at 257 nm (E, 33); itsinfra-red spectrum (determined with a mixture with potassium bromide)shows principal absorption bands as follows:

LII

shoulder, 2,840 shoulder, 1,735 strong, 1,715 medium, 1,630 medium,1,570 medium, 1,450 strong, 1,403 medium, 1,380 strong, 1,350 medium,1,335 shoulder, 1,305 shoulder, 1,248 strong, 1,220 weak, 1,192 medium,1,165 medium, 1,122 strong, 1,090 strong, 1,060 strong, 1,035 verystrong, 975 medium, 942 medium, 920 shoulder, 900 weak, 865 medium, 848weak, 828 weak, 815 weak, 775 medium, 730 medium, 690 weak, 678 veryweak, 655 weak, 630 shoulder, 612 weak, 570 weak, 548 weak, 525 veryweak, 510 very weak; and its optical rotation is [a],, 3 3 (0 0.5, 0.1NNaOH), 1011 7 2*: 3 (0 0.5, 0.1N NaOH), 1011 +20 3 (0 0.5, 0.1N NaOH thepremix containing 0.05 to 20% by weight of said 21,190 RP.

4. A concentrate for addition to animal feedstuff comprising, inassociation with a physiologically innoc uous carrier or an edibledenaturant, 20% to 99.9% by weight of said concentrate of the antibioticherein designated 21,190 RP which possesses the followingcharacteristics: it is a white crystalline powder, melting at 226-228C.,soluble in water at pH 9 and in dimethylformamide, sparingly soluble inmethanol and ethanol, and practically insoluble in hexane and benzene;it contains carbon, hydrogen, oxygen and chlorine and has the elementarycomposition: C 50.5%, H 667:, O 38.4%, C1 45%; its ultra-violet spectrumin aqueous 0.1N sodium hydroxide solution shows an absorption maximum at292 nm (E, 59) and an absorption minimum at 257 nm (E, 33); itsinfra-red spectrum (determined with a mixture with potassium bromide)shows principal absorption bands as follows: 3,440 strong, 2,970 medium,2,930 strong, 2,900 shoulder, 2,840 shoulder, 1,735 strong, 1,715medium, 1,630 medium, 1,570 medium, 1,450 strong, 1,403 medium, 1,380strong, 1,350 medium, 1,335 shoulder, 1,305 shoulder, 1,248 strong,1,220 weak, 1,192 medium, 1,165 medium, 1,122 strong, 1,090 strong,1,060 strong, 1,035 very strong, 975 medium, 942 medium, 920 shoulder,900 weak, 865 medium, 848 weak, 828 weak, 815 weak, 775 medium, 730medium, 690 weak, 678 very weak, 655 weak, 630 shoulder, 612 weak, 570weak, 548 weak, 525 very weak, 510 very weak; and its optical rotationis [01],, 3 I 3 (c 0.5. 0.1N NaOH), 1011 7 2 3 (c 0.5, 0.1N NaOH), [a]20 1 3 (0 0.5.0.11 1 NaOH).

5. A method of promoting the growth of animals which comprises feedingto the animals a feedstuff comprising a growth-promoting amount of theantibiotic herein designated 21,7190 RP, which possesses the followingcharacteristics: it is a white crystalline pow der, melting at226-228C., soluble in water at pH 9 and in dimethylformamide, sparinglysoluble in methanol and ethanol, and practically insoluble in hexane andbenzene; it contains carbon, hydrogen, oxygen and chlorine and has theelementary composition: C 50.5% H 6.6% O 38.4% C1 4.5%, its ultra-violetspectrum in aqueous 0.1N sodium hydroxide solution shows an absorptionmaximum at 292 nm (E, 59) and an absorption minimum at 257 nm (E, 33 1;its infra-red spectrum (determined with a mixture with potassiumbromide) shows principal absorption hands as follows: 3,440 strong,2,970 medium, 2,930 strong. 2,900 shoulder, 2,840 shoulder, 1,735strong, 1,715 medium, 1,630 medium, 1,570 medium, 1,450 strong, 1,403medium, 1,380 strong, 1,350 medium, 1,335 shoulder, 1,305 shoulder,1,248 strong, 1,220 weak, 1,192 medium, 1,165 medium, 1,122 strong,1,090 strong, 1,060 strong, 1,035 very strong, 975 medium, 942 medium,920 shoulder, 900 weak, 865 medium, 848 weak, 828 weak, 815 weak, 775medium, 730 medium, 690 weak, 678 very weak, 655 weak, 630 shoulder, 612weak, 570 weal-c, 548 weak, 525 very weak, 510 very weak; and itsoptical rotation is 1041 +3 i 3 (c 0,5, 0.1N NaOH), {M 7 i 3 lc'=0.5,0.11\l NuOH), [a];,,,-,-, =+20i 3 05, 0.1N NaOH),

6. A method for the treatment of bacterial infections in domesticanimals which comprises administering to the animals parenterally anantibiotically effective amount of the antibiotic herein designated21,190 RP. which possesses the following characteristics: it is a whitecrystalline powder, melting at 226-228C., soluble in water at pH 9 andin dimethylformamide, sparingly soluble in methanol and ethanol, andpractically insoluble in hexane and benzene; it contains carbon,hydrogen, oxygen and chlorine and has the elementary composition: C50.5% H 66% O 38.4% C] 4.5%; its ultraviolet spectrum in aqueous 0.1Nsodium hydroxide solution shows an absorption maximum at 292 nm (E, =59)and an absorption minimum at 257 nm (E 33); its infra-red spectrum(determined with a mixture with potassium bromide) shows principalabsorption bands as follows: 3,440 strong, 2,970 medium, 2,930 strong,2,900 shoulder, 2,840 shoulder, 1,735 strong, 1,715 medium, 1,630medium, 1,570 medium, 1,450 strong, 1,403 medium, 1,380 strong, 1,350medium, 1,335 shoulder, 1,305 shoulder, 1,248 strong, 1,220 weak, 1,192medium, 1,165 medium, 1,122 strong, 1,090 strong, 1,060 strong, 1,035very strong, 975 medium, 942 medium, 920 shoulder, 900 weak, 865 medium,848 weak, 828 weak, 815 weak, 775 medium, 730 medium, 690 weak, 678 veryweak, 655 weak, 630 shoulder, 612 weak, 570 weak, 548 weak, 525 veryweak. 510 very weak; and its optical rotation is 1a],, 3 3 (0 0.1, 0.1NNaOH), 1011 7i 3 (c 0.5, 0.1N NaOH), 101M 20 3 (c 0.5. 0.1N NaOH).

1. A FEEDSTUFF COMPOSITION COMPRISING AN ANIMAL FEEDSTUFF AND 0.1 TO0.0001% BY WEIGHT OF SAID FEEDSTUFF OF THE ANTIBIOTIC HEREIN DESIGNATED21, 190RP, WHICH POSSESSES THE FOLLOING CHARACTERISTICS: IT IS A WHITECRYSTALLINE POWDER, MELTING A T 226*-228*C., SOLUBLE IN WATER AT PH9 ANDIN DIMETHYLFORMAMIDE, SPARINGLY SOLUBLE IN METHANOL AND ETHANOL, ANDPREACTICALLY INSOLUBLE IN HEXANE AND BENZENE, IT CONTAINS CARBON,HYDROGEN, OXYGEN AND CHLORINE AND HAS THE ELEMENTARY COMPOSITION:C=50.5%''=6.6% 0= 38.4% "1= 4.5%, ITS ULTRA-VIOLET SPECTRUM IN AQUEOUS0.1N SODIUM HYDROXIDE SOLUTION SHOWS AN ABSORPTION MAXIMUM AT 292NM(E1CM1%=33) AND AN ABSORPTION MINIMUM AT 257NM (E1CM1%=33), ITSINFRA-RED SPECTRUM (DETERMINED WITH A MIXTURE WITH POTASSIUM BROMIDE)SHOWS PRINCIPAL ABSORPTION BANDS AS FOLLOWS: 3,440 STRONG, 2,970 MEDIUM,2,9300 STRONG SHOULDER, 2,840 SHOULDER, 1,735 STRONG, 1,715 MEDIUM,1,630 MEDIUM, 1,570 MEDIUM, 1,335 SHOULDER8 1,305 SHOULDER, 1,248STRONG, 1,220 WEAK, 1,192 MEDIUM, 1,165 MEDIUM, 1,122 STRONG, 1,090STRONG, 1,060 STRONG, 1,035 VERY STRONG, 975 MEDIUM, 942 MEDIUM, 920SHOULDER, 900 WEAK, 865 MEDIUM, 848 WEAK, 828 WEAK, 815 WEAK, 775,MEDIUM730 MEDIUM, 690 WEAK, 678 VERY WEAK, 655 WEAK 630 SHOULDER, 612 WEAK,570 WEAK, 548 WEAK, 525 VERY WEAK, 510 VERY WEAK, AND ITS OPTICALROTATION IS (A)D20=+3*$3*(C=0.5,0.1N NAOH),(A)43620= +7*+3*(C=0.5, 0.1NNAOH), (A)365**20=20$+3*(A=0,5,
 2. A feedstuff according to claim 1,comprising 1 to 80 g. of 21.190 RP per metric ton of said feedstuff. 3.A premix for an animal feedstuff which comprises, in association with ananimal feedstuff or a physiologically innocuous carrier, the antibioticherein designated 21,190 RP which possesses the followingcharacteristics: it is a white crystalline powder, melting at226*-228*C., soluble in water at pH 9 and in dimethylformamide,sparingly soluble in methanol and ethanol, and practically insoluble inhexane and benzene; it contains carbon, hydrogen, oxygen and chlorineand has the elementary composition: C 50.5% H 6.6% O 38.4% Cl 4.5%; itsultra-violet spectrum in aqueous 0.1N sodium hydroxide solution shows anabsorptIon maximum at 292 nm (E1 cm1% 59) and an absorption minimum at257 nm (E1 cm1% 33); its infra-red spectrum (determined with a mixturewith potassium bromide) shows principal absorption bands as follows:3,440 strong, 2,970 medium, 2,930 strong, 2,900 shoulder, 2,840shoulder, 1,735 strong, 1,715 medium, 1,630 medium, 1,570 medium, 1,450strong, 1,403 medium, 1,380 strong, 1,350 medium, 1,335 shoulder, 1,305shoulder, 1,248 strong, 1,220 weak, 1,192 medium, 1,165 medium, 1,122strong, 1,090 strong, 1,060 strong, 1,035 very strong, 975 medium, 942medium, 920 shoulder, 900 weak, 865 medium, 848 weak, 828 weak, 815weak, 775 medium, 730 medium, 690 weak, 678 very weak, 655 weak, 630shoulder, 612 weak, 570 weak, 548 weak, 525 very weak, 510 very weak;and its optical rotation is ( Alpha )D20 + 3* + or - 3* (c 0.5, 0.1NNaOH), ( Alpha )43620 + 7* + or - 3* (c 0.5, 0.1N NaOH), ( Alpha )36520+20* + or - 3* (c 0.5, 0.1N NaOH); the premix containing 0.05 to 20% byweight of said 21,190 RP.
 4. A concentrate for addition to animalfeedstuff comprising, in association with a physiologically innocuouscarrier or an edible denaturant, 20% to 99.9% by weight of saidconcentrate of the antibiotic herein designated 21,190 RP whichpossesses the following characteristics: it is a white crystallinepowder, melting at 226*-228*C., soluble in water at pH 9 and indimethylformamide, sparingly soluble in methanol and ethanol, andpractically insoluble in hexane and benzene; it contains carbon,hydrogen, oxygen and chlorine and has the elementary composition: C50.5%, H 6.6%, O 38.4%, Cl 4.5%; its ultra-violet spectrum in aqueous0.1N sodium hydroxide solution shows an absorption maximum at 292 nm (E1cm1% 59) and an absorption minimum at 257 nm (E1 cm1% 33); its infra-redspectrum (determined with a mixture with potassium bromide) showsprincipal absorption bands as follows: 3,440 strong, 2,970 medium, 2,930strong, 2,900 shoulder, 2,840 shoulder, 1,735 strong, 1,715 medium,1,630 medium, 1,570 medium, 1,450 strong, 1,403 medium, 1,380 strong,1,350 medium, 1,335 shoulder, 1,305 shoulder, 1,248 strong, 1,220 weak,1,192 medium, 1,165 medium, 1,122 strong, 1,090 strong, 1,060 strong,1,035 very strong, 975 medium, 942 medium, 920 shoulder, 900 weak, 865medium, 848 weak, 828 weak, 815 weak, 775 medium, 730 medium, 690 weak,678 very weak, 655 weak, 630 shoulder, 612 weak, 570 weak, 548 weak, 525very weak, 510 very weak; and its optical rotation is ( Alpha )D20 3* +or - 3* (c 0.5, 0.1N NaOH), ( Alpha )43620 + 7* + or - 3* (c 0.5, 0.1NNaOH), ( Alpha )36520 + 20* + or - 3* (c 0.5, 0.1N NaOH).
 5. A method ofpromoting the growth of animals which comprises feeding to the animals afeedstuff comprising a growth-promoting amount of the antibiotic hereindesignated 21,7190 RP, which possesses the following characteristics: itis a white crystalline powder, melting at 226*-228*C., soluble in waterat pH 9 and in dimethylformamide, sparingly soluble in methanol andethanol, and practically insoluble in hexane and benzene; it containscarbon, hydrogen, oxygen and chlorine and has the elementarycomposition: C 50.5% H 6.6% O 38.4% Cl 4.5%; its ultra-violet spectrumin aqueous 0.1N sodium hydroxide solution shows an absorption maximum at292 nm (E1 cm1% 59) and an absorption minimum at 257 nm (E1 cm1% 33);its infra-red spectrum (determined with a mixture with potassiumbromide) shows principal absorption bands as follows: 3,440 strong,2,970 medium, 2,930 strong, 2,900 shoulder, 2,840 shoulder, 1,735strong, 1,715 medium, 1,630 medium, 1,570 medium, 1,450 strong, 1,403medium, 1,380 strong, 1,350 medium, 1,335 shoulder, 1,305 shoulder,1,248 strong, 1,220 weak, 1,192 medium, 1,165 medium, 1,122 strong,1,090 strong, 1,060 strong, 1,035 very strong, 975 medium, 942 medium,920 shoulder, 900 weak, 865 medium, 848 weak, 828 weak, 815 weak, 775medium, 730 medium, 690 weak, 678 very weak, 655 weak, 630 shoulder, 612weak, 570 weak, 548 weak, 525 very weak, 510 very weak; and its opticalrotation is ( Alpha )D20 +3* + or - 3* (c 0.5, 0.1N NaOH), ( Alpha)43620 + 7* + or - 3* (c 0.5, 0.1N NaOH), ( Alpha )36520 + 20* + or -3*(c 0.5, 0.1N NaOH).
 6. A method for the treatment of bacterialinfections in domestic animals which comprises administering to theanimals parenterally an antibiotically effective amount of theantibiotic herein designated 21,190 RP, which possesses the followingcharacteristics: it is a white crystalline powder, melting at226*-228*C., soluble in water at pH 9 and in dimethylformamide,sparingly soluble in methanol and ethanol, and practically insoluble inhexane and benzene; it contains carbon, hydrogen, oxygen and chlorineand has the elementary composition: C 50.5% H 6.6% O 38.4% Cl 4.5%; itsultra-violet spectrum in aqueous 0.1N sodium hydroxide solution shows anabsorption maximum at 292 nm (E1 cm1% 59) and an absorption minimum at257 nm (E1 cm1% 33); its infra-red spectrum (determined with a mixturewith potassium bromide) shows principal absorption bands as follows:3,440 strong, 2,970 medium, 2,930 strong, 2,900 shoulder, 2,840shoulder, 1,735 strong, 1,715 medium, 1,630 medium, 1,570 medium, 1,450strong, 1,403 medium, 1,380 strong, 1,350 medium, 1,335 shoulder, 1,305shoulder, 1,248 strong, 1,220 weak, 1,192 medium, 1,165 medium, 1,122strong, 1,090 strong, 1, 060 strong, 1,035 very strong, 975 medium, 942medium, 920 shoulder, 900 weak, 865 medium, 848 weak, 828 weak, 815weak, 775 medium, 730 medium, 690 weak, 678 very weak, 655 weak, 630shoulder, 612 weak, 570 weak, 548 weak, 525 very weak, 510 very weak;and its optical rotation is ( Alpha )D20 + 3* + or - 3* (c 0.1, 0.1NNaOH), ( Alpha )43620 + 7* + or - 3* (c 0.5, 0.1N NaOH), ( Alpha)36520 + 20* + or - 3* (c 0.5, 0.1N NaOH).